Abstract

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

eLife digest

Calcium ions regulate many processes in the human body. The calcium-sensing receptor, called CaSR, is responsible for maintaining a stable level of calcium ions in the blood. This receptor can detect small changes in the concentration of calcium ions, and activates signalling events within the cell to restore the level of calcium ions back to normal. Abnormal activity of this receptor is associated with severe diseases in humans

CaSR is found in the surface membrane of cells and belongs to a family of proteins called G-protein coupled receptors. Much of the protein extends out of the cell and interacts with calcium ions, phosphate ions and certain other molecules such as amino acids. However, it was not well understood how these small molecules bind to CaSR and how this activates the receptor.

Geng et al. have now used a technique called X-ray crystallography to view the three-dimensional structure of the exterior domain of CaSR in its resting state and active state. These structures revealed that, contrary to expectations, calcium ions are not the main activator of the receptor. Instead, Geng et al. found that CaSR adopts an inactive state in the absence or presence of calcium ions, while the active state only forms when an amino acid is bound.

Furthermore investigation showed that calcium ions are needed to stabilise the active form, while phosphate ions keep the inactive form stable. Geng et al. also identified the shape changes that must occur as CaSR transitions from its inactive to its active state. In particular, an amino acid binding to the exterior domain causes it to close like a venus flytrap, which is a crucial step in activating the receptor.

Taken together, the findings show that the amino acids and calcium ions act jointly to fully activate CaSR. The next steps are to determine the structure of the entire receptor with and without its small molecule partners and to use these structures to design drugs that can alter CaSR’s activity in order to treat human diseases.

Structural information for class C GPCRs is available for the extracellular domains (ECD) of mGluRs (Kunishima et al., 2000; Muto et al., 2007; Tsuchiya et al., 2002) and GABAB receptor (Geng et al., 2013, 2012), as well as the transmembrane domains of mGluRs (Dore et al., 2014; Wu et al., 2014). Here, we present the first crystal structures of the entire extracellular domain of human CaSR in two different functional states. These structures reveal novel binding sites for Ca2+, PO43- and L-Trp, identify L-Trp as an agonist of the receptor, and demonstrate that these ions and amino acids collectively control the function of CaSR.

Results

Structure of CaSR ECD homodimer

The ECD of human CaSR was secreted from baculovirus-infected insect cells as a disulfide-tethered homodimer (Figure 1—figure supplement 1). It contains 11 potential N-linked glycosylation sites. Disruption of three of the glycosylation sites did not alter CaSR signaling (Figure 1—figure supplement 1). Formation of well-diffracting crystals required partial deglycosylation of the receptor through mutation and enzymatic digestion. We obtained two different forms of CaSR ECD crystals. Form I was crystallized in the absence and presence of 2 mM Ca2+, and form II in the presence of 10 mM L-Trp and 10 mM Ca2+ (Table 1).

In both CaSR ECD structures, the two protomers interact in a side-by-side fashion while facing opposite directions (Figure 1; Figure 1—figure supplement 2). Each CaSR ECD protomer consists of three domains, LB1, LB2 and CR. The two lobe-shaped domains LB1 and LB2 form a VFT module similar to that of mGluRs (Kunishima et al., 2000; Muto et al., 2007; Tsuchiya et al., 2002) and GABAB receptor (Geng et al., 2013, 2012). The relative orientation between the LB2 and CR domains is fixed through an interdomain disulfide linkage (C236-C561), and the CR domain is positioned to amplify and transmit the conformational variations within the VFT module.

Crystal structures of human CaSR ECD.

(A) Inactive-state structure of CaSR ECD homodimer in the presence of 2 mM Ca2+. (B) Active-state structure of CaSR ECD homodimer in the presence of 10 mM Ca2+ and 10 mM L-Trp. Each structure is shown in cartoon (front view) or surface (side view) representations that are related by a 90°-rotation about the vertical axis. Each protomer is colored according to its individual domains (LB1, light blue; LB2, blue; CR, purple). The various ligands (L-Trp, Ca2+, PO43-, SO42-) are displayed as space-filling models. Observed carbohydrates are shown as ball-and-stick models in gray. Disulfide bridges are in yellow.

Form I crystal structure of CaSR ECD represents the inactive configuration since the VFT modules of both protomers are in the open conformation associated with the resting state (open-open), and the interdomain cleft is empty. In addition, each protomer structure contains one Ca2+ ion and three SO42- ions (Figure 1A).

In the form II crystal structure, both protomers of CaSR ECD have the closed conformation associated with agonist binding (closed-closed). Surprisingly, the ligand-binding cleft of each protomer is solely occupied by an L-Trp molecule. Ca2+ is bound at four novel sites in the CaSR ECD structure, including one at the homodimer interface. Each CaSR ECD molecule also contains two PO43- ions (Figure 1B).

Agonist binding induces large conformational changes within the CaSR ECD homodimer. First, the VFT module of each protomer undergoes domain closure. Alignment based on the LB1 domains showed that the LB2 domains of inactive and active structures are related by a 29° rotation (Figure 2A). Second, the LB2 domains of the two protomers approach each other, resulting in an expansion of the homodimer interactions involving LB2 domains. Third, the CR domains of the two subunits interact to form a large homodimer interface that is unique to the active state. The CR domains are brought into close contact by the motion involving LB2 since the two domains are rigidly associated within each subunit. Finally, the structural reorganization of CR domains reduces the distance between the C-termini of the two subunits from 83 Å to 23 Å (Figure 2B,C). This CR domain movement may cause reorientation of the transmembrane domains during receptor activation.

Agonist-induced conformational changes.

(A) Superposition of the inactive (orange) and active (blue) CaSR ECD structures based on the LB1 domain of one protomer (front view, left; side view, right). Green line is the axis of rotation that relates the LB2 domains of the superimposed protomers (rotation χ = 29°, screw translation τχ = −2.2 Å). (B, C) Surface representation of inactive (B) and active (C) structures in front (top) and bottom (bottom) views. Distance between C-termini of the two subunits (yellow) is marked by dashed line for each homodimer.

The LB1-LB1 dimer interface buries over 3800 Å2 of solvent-accessible surface area, and can be divided into two regions. Site I is located at the center of LB1 domain and is flanked on either side by the two symmetric parts of site II (Figure 3A,B).

The dimer interactions at site I of CaSR are predominantly hydrophobic and involve tightly packed leucine and phenylalanine residues (L112, L156, L159, and F160). The disease-causing mutation L159P renders the receptor less sensitive to Ca2+ (Grant et al., 2011; Hendy et al., 2009) (Figure 3C). In addition, site I features an inter-subunit disulfide bridge located at the tip of helix C.

Site II is unique to the CaSR ECD structures. It involves an arm-like long loop stretched out from one subunit to reach its binding partner (Figure 3A,B; Figure 3—figure supplement 1). The dimer interactions at site II include hydrogen bonds and hydrophobic contacts. Several disease-causing mutations are located at this interface (S53P, P55L, and Y161C) (Hendy et al., 2009). Substitution of a deeply buried interfacial residue W458 with alanine also decreased the potency of Ca2+ (Figure 3C). These observations indicate that formation of a stable homodimer is important for CaSR function.

Agonist-induced homodimer interface

Agonist binding causes an expansion of the dimer interactions involving LB2 domain. In the inactive homodimer, only minimal contacts occur between the LB2 domains (Figure 3A). In the active state, LB2 of one protomer interacts with all three domains of a second protomer (Figure 3B). These contacts are predominantly hydrophilic, and bury 1000 Å2 of solvent accessible surface area.

LB2 mediates dimer interactions primarily through a central helix (G) that transverses the domain (Figure 3B). First, the top of helix G contacts LB1 domain of the opposing subunit through two symmetric salt bridges between D215 and R172 (site III). Second, the LB2-LB2 contacts involve the midsection of helix G, and feature a hydrogen bond between R227 and S240, and water-mediated contacts by E224 (site IV). Finally, LB2 interacts with the CR domain of a second subunit through a Ca2+ ion (site V). This Ca2+ ion bridges the end of helix G in LB2 with a loop in CR domain. An abundance of disease-related mutations are found at these dimer interaction sites, including (1) R172G and D215G at site III, (2) R227L and R227Q at site IV, and (3) G557E at site V (Hendy et al., 2009). All these mutations disrupt agonist-dependent dimer contacts, and decreased agonist efficacy (Heath et al., 1996; Hendy et al., 2009; Wystrychowski et al., 2005) (Figure 3C).

Agonist binding also induces the formation of a novel homodimer interface between the CR domains that covers approximately 1200 Å2 of solvent accessible surface area (site VI) (Figure 3B). The CR-CR interactions are mediated by two β-strands and their connecting loop from each subunit. Key contacts include two cross-subunit hydrogen bonds (T560, E558), hydrophobic contacts (I554, P569), and electrostatic interactions (R551). Among these, R551K is a known disease-causing mutation that reduced the receptor response (Hendy et al., 2009; Toke et al., 2007) (Figure 3C).

Amino acid recognition

In the active structure, the amino acid L-Trp is bound at the interdomain cleft of the VFT module, in agreement with previous mutational data (Mun et al., 2005; Mun et al., 2004; Zhang et al., 2014, 2002) (Figure 4A; Figure 4—figure supplement 1). L-Trp facilitates extracellular domain closure of CaSR by contacting both LB1 and LB2 domains of the VFT module (Figure 4B,C; Figure 4—figure supplement 1). The interactions between CaSR ECD and L-Trp are primarily mediated by hydrogen bonds. (1) The carboxylic acid group of L-Trp forms hydrogen bonds through both oxygen atoms with LB1 residues S147, A168, and S170. (2) The backbone nitrogen of L-Trp is hydrogen-bonded to A168 and S170 of LB1 domain. (3) The indole nitrogen of L-Trp forms two hydrogen bonds with E297 of LB2 domain. (4) L-Trp is engaged in hydrophobic contacts with both LB1 and LB2 residues including W70, T145, Y218, and A298. The extensive contacts between backbone atoms of L-Trp and the receptor suggest that other amino acids may bind to CaSR in a similar fashion to induce domain closure.

We measured the direct interaction between L-Trp and CaSR ECD by scintillation proximity assay (SPA) (Quick and Javitch, 2007) (Figure 4D). CaSR ECD exhibited binding of [3H]-L-Trp in the absence and presence of Ca2+. Isotope dilution of [3H]-L-Trp with nonradioactive L-Trp led to a displacement of [3H]-L-Trp in a concentration-dependent manner. The addition of 2 mM Ca2+ increased the amount of L-Trp bound to CaSR ECD at any given concentration, suggesting that extracellular Ca2+ enhances L-Trp binding, possibly by affecting the L-Trp-binding affinity and kinetics of CaSR. The half-maximal inhibitory concentration of L-Trp (IC50, concentration at which 50% displacement of [3H]-L-Trp was observed) was approximately 2 mM regardless of whether the experiment was performed in the absence (2.11 ± 0.72 mM) or presence (2.04 ± 0.10 mM) of 2 mM Ca2+. Nevertheless, the binding affinity of L-Trp to CaSR ECD with and without Ca2+ may still differ as it depends on the concentration and binding affinity of the radiolabeled ligand. Further studies are needed to characterize the effect of Ca2+ on L-amino acid binding at the orthosteric agonist site of CaSR.

We found that L-Trp directly stimulated intracellular Ca2+ mobilization through CaSR (Figure 4E,F), in agreement with previous findings (Conigrave et al., 2004, 2000; Rey et al., 2005; Young and Rozengurt, 2002). L-Trp-induced CaSR activation required the presence of extracellular Ca2+ above a threshold level of 0.5 mM. The effect of L-Trp on CaSR was concentration-dependent, with an apparent half-maximal effective concentration (EC50) of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy and potency of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR (Conigrave et al., 2000, 2004).

Ca2+-binding

We identified four distinct Ca2+-binding sites within each protomer of the active structure using anomalous difference maps, and named these sites 1 through 4 (or 1′-4’ in the second protomer) (Figure 5A,B; Figure 5—figure supplement 1; Table 1—source data 1). The inactive structure revealed electron density at site 2 that is consistent with a bound Ca2+ ion (Figure 5C,D). None of the Ca2+-binding sites observed in the CaSR ECD structures has been reported previously.

Site 1 is located in a loop region at the top of LB1 domain (Figure 5A,B). The bound Ca2+ ion is primarily coordinated by backbone carbonyl oxygen atoms of I81, S84, L87, and L88 (site 1′). The structural configuration of site 1 is similar in the inactive and active structures even though it is only occupied in the active state (Figure 5—figure supplement 1). The disease-causing mutation I81M (Hendy et al., 2009) is located at site 1, and it abolished Ca2+-dependent receptor response, possibly by disrupting a tightly packed hydrophobic patch adjacent to Ca2+-binding site 1 (Figure 6A,B). This implies that the local conformation of this loop region is important for receptor function, and the Ca2+ ion stabilizes the observed conformation in the active state.

Mutational analysis of Ca2+-binding sites.

(A, B) Dose-dependent Ca2+-stimulated IP accumulation (A) and intracellular Ca2+ mobilization (B) in cells transiently expressing wt or mutant CaSR. Naturally occurring inactivating mutations I81M and T100I are located at various Ca2+-binding sites. The single mutation N102I was designed based on structure to interfere with Ca2+-binding.

The inactive and active structures share a common Ca2+-binding mode at site 2, suggesting that the bound Ca2+ is an integral part of the CaSR structure (Figure 5A–D; Figure 5—figure supplement 1). Site 2 is positioned directly above the interdomain crevice in LB1 domain, and it abuts the L-Trp binding site in the cleft. The Ca2+ ion is coordinated by the hydroxyl group of T100 in both states, and by the carboxyl group of N102 through a water molecule in the active structure. In addition, T145, another residue lining the site, forms part of the L-Trp binding cleft in the active state. Therefore, an intact Ca2+ site 2 provides an essential framework for L-Trp recognition. Indeed, introducing a hydrophobic residue at this site through mutations T100I, N102I, or T145I nearly eliminated Ca2+-induced receptor activity (Figure 4D; Figure 6A,B).

Site 3 is positioned at the edge of the interdomain cleft in LB2 domain (Figure 5A,B). The Ca2+ ion is coordinated by the hydroxyl groups of S302 and S303 either directly (site 3′) or indirectly through water molecules. Alignment of the inactive and active structures in this region showed a small conformational change (Figure 5—figure supplement 1). Ca2+ ion stabilizes a loop conformation that permits an interdomain hydrogen bond between the neighboring LB2 residue S301 and LB1 residue R66. Such interaction enhances domain closure of CaSR ECD for receptor activation.

Among all four Ca2+-binding sites in CaSR ECD structure, site 4 is most closely associated with receptor activation because it directly participates in the formation of an active receptor conformation. Site 4 is part of the homodimer interface formed upon agonist binding, bridging the LB2 domain of one subunit and CR domain of the second subunit (Figure 5A,B). The Ca2+ ion is coordinated by three interfacial residues, including the carboxylate group of D234 and carbonyl oxygen of E231 and G557 (Figure 5B). The natural mutation G557E (Hendy et al., 2009) reduced the potency of Ca2+ possibly by affecting backbone conformation, thereby weakening the affinity of Ca2+ for this site (Figure 6A,B). Our structural data indicate that the Ca2+ ion at site 4 stabilizes the active conformation of the receptor by facilitating homodimer interactions between the membrane-proximal LB2 and CR domains.

The Ca2+ ions have different peak heights in the anomalous difference maps, which are correlated with different Ca2+-occupancies at various sites. The Ca2+ ions at sites 1 and 2 have strong peaks (12.1–13.1 σ), indicating that these are high-occupancy sites. The Ca2+ ions at sites 3 (7.3 – 9.0 σ) and 4 (5.8 σ) have weaker anomalous peaks, which suggest low occupancy and possibly low affinity. The Ca2+ ion at site 4 has the weakest peak compared with other sites, which is consistent with the site being occupied only at elevated Ca2+ concentration for receptor activation.

Anion binding

We identified a total of four anion-binding sites in the inactive and active CaSR ECD structures based on anomalous difference maps (1–4 or 1'-4’ in the second protomer) (Figure 7; Figure 7—figure supplement 1; Table 1—source data 1). Sites 1–3 are located above the interdomain cleft in the LB1 domain, and site 4 is part of LB2 domain.

In the inactive structure, electron densities revealed that anions were bound at sites 1–3 (Figure 7A,B). In the active structure, only sites 2 and 4 are occupied (Figure 7C,D). We modeled the anions as SO42- ions in the inactive state and PO43- ions in the active state given their respective presence in the crystallization reagents. It is also possible that endogenous anions other than SO42- and PO43- are bound at these sites.

The anion at site 1 is coordinated by the guanidine group of R62 and backbone nitrogen of Y63 (Figure 7B). In the absence of a bound anion in the active state, the side chain of LB1 residue R62 reaches across the interdomain cleft to form a salt bridge with E277 of LB2 domain (Figure 7E). This contact stabilizes the closed conformation of the CaSR VFT module.

The anion bound at site 2 is held in place by multiple hydrogen bonds with the side chains of R66, R69, W70, and S417, and main chains of R415, I416, and S417 (Figure 7B,D). The structural integrity of this site is important for receptor function. Each of the mutations R66H, R69E, and S417L essentially eradicated receptor signaling (Figure 8A,B). Among these, R66H is a disease-associated mutation (Hendy et al., 2009; Pidasheva et al., 2006).

Anion-binding site 3 is adjacent to site 2. The bound anion is also coordinated by R66 and additionally by T412 (Figure 7B). In the active state, the side chain of LB1 residue R66 forms a hydrogen bond with S301 of LB2 domain (Figure 7E). This interaction also serves to maintain the CaSR ECD in a closed conformation.

The anion at site 4 is coordinated by H192, T195, K225 and R520 in the active structure (Figure 7D). In the absence of a bound anion, the side chains of several binding-site residues are disordered in the inactive structure. This indicates that the anion serves to stabilize the local conformation of the receptor structure.

We measured the effect of anion on Ca2+-dependent receptor response and found that the presence of SO42- slightly decreased receptor activity, increasing the EC50 of Ca2+ by approximately 25% (Figure 8C). This finding confirmed that anions have a negative allosteric effect on the receptor.

Discussion

Implications for agonist-dependent receptor activation

Our structural analyses of CaSR ECD provide direct evidence that amino acids are agonists of CaSR, and they act concertedly with Ca2+ to achieve full receptor activation. L-Trp, the amino acid used in this study, fits the role of an orthosteric agonist for CaSR. (1) It binds at the interdomain crevice of the VFT module, the canonical agonist-binding site for class C GPCRs (Geng et al., 2013; Kunishima et al., 2000; Muto et al., 2007; Tsuchiya et al., 2002). (2) L-Trp shares a common receptor-binding mode with the endogenous agonists of mGluRs and GABAB receptor, which are also amino acids or their analogs (Geng et al., 2013; Kunishima et al., 2000; Muto et al., 2007; Tsuchiya et al., 2002). The residues involved in agonist recognition are located at the same positions in the structures of these receptors (Figure 4—figure supplement 1). For example, a conserved serine residue is responsible for securing the carboxylate of L-Trp, glutamate and GABA in CaSR (S147), mGluRs (S165) (Kunishima et al., 2000) and GABAB receptor (S130) (Geng et al., 2013), respectively. (3) L-Trp interacts with both LB1 and LB2 domains to facilitate extracellular domain closure, a crucial first step during CaSR activation. In contrast, no Ca2+ ion is found at the putative orthosteric agonist-binding site to induce domain closure. (4) Mutations of L-Trp-binding residues completely blocked Ca2+-induced IP accumulation and intracellular Ca2+ mobilization (Silve et al., 2005; Zhang et al., 2002), indicating that L-Trp is required for Ca2+-mediated receptor response. (6) L-Trp directly activates CaSR-mediated intracellular Ca2+ mobilization in the presence of extracellular Ca2+.

On the other hand, Ca2+ ion fulfills at least three functional roles. First, it maintains the structural integrity of CaSR, as manifested by the importance of Ca2+-binding site 2 for receptor function. Second, it is directly involved in receptor activation. Specifically, the Ca2+ ion at site 4 stabilizes the unique homodimer interface between membrane-proximal LB2 and CR domains in the active state. Third, Ca2+ enhances L-Trp binding to CaSR ECD, possibly by reinforcing the L-Trp bound and active conformation of the receptor. Furthermore, the actions of Ca2+ and amino acids on CaSR are inter-dependent. While the presence of extracellular Ca2+ above a threshold level is required for amino-acid-mediated CaSR activation, amino acids increase the sensitivity of the receptor toward Ca2+. Taken together, we conclude that amino acids and Ca2+ ions are indeed co-agonists of CaSR, acting jointly to trigger receptor activation.

This then led us to an intriguing question: How does Ca2+ ion activate the receptor on its own as demonstrated in various cell-based functional assays? It is possible that amino acids are present in cell culture media and extracellular fluid at sufficient concentration to prime the receptor to respond to increasing concentrations of Ca2+. We have obtained crystals of CaSR ECD in the absence of any additional amino acids. Nevertheless, the structure showed a stretch of continuous density in the interdomain cleft that may belong to an endogenous ligand (Figure 4—figure supplement 2; Table 1—source data 2). Despite multiple attempts, we have not been able to identify the structure of this endogenous ligand through mass spectrometry. In light of the L-Trp-bound CaSR ECD structure, we reason that the endogenous ligand may be an amino acid or even a mixture of amino acids. In the inactive structure, the endogenous ligand was likely removed by citrate buffer (pH 5.5) during enzymatic deglycosylation.

Metabolic balances of Ca2+ and PO43- are linked through hormonal factors such as parathyroid hormone and fibroblast growth factor-23, which control the homeostasis of both ions (Brown, 2013; Quinn et al., 2013; Tyler Miller, 2013). We therefore reason that the physiologically relevant anion bound to CaSR is likely PO43-, which primarily exists in a HPO42-/H2PO4- mixture in biological systems. First, the anion at site 2 is required for structural stability of the receptor. Second, the anions at sites 1 and 3 appear to stabilize the inactive conformation. Binding of anions at these sites prevent favorable interactions across the interdomain cleft that promote VFT closure. Third, the presence of anion decreases CaSR-mediated IP accumulation. Therefore, these anions may exert a negative modulatory effect on CaSR activity.

The presence of anion-binding sites in CaSR may also provide a mechanism for CaSR to sense polycations such as polyamines. Increasing concentrations of polyamines could potentially compete with arginine residues at the anion-binding sites to bind to PO43-, thereby prompting the dissociation of PO43- from relatively weak sites, and releasing their inhibitory effect on the receptor. This would drive CaSR toward its active-state conformation. Our hypothesis would predict that polycations with higher number of positive charges will be more effective agonists. Indeed, previous studies have shown that polyamines mediate an increase in intracellular inositol phosphate and Ca2+ accumulation with the rank order seprmine > spermidine > putrescine (Cheng et al., 2004; Quinn et al., 1997).

In summary, activation of CaSR involves an intricate interplay of amino acids, Ca2+, and possibly PO43- ions. Like other GPCRs (Rosenbaum et al., 2011), CaSR exists in a conformational equilibrium between inactive and active states (Figure 9). (1) CaSR adopts an open conformation in the resting state, and PO43- ions promote the inactive configuration. (2) An L-amino acid closes the groove in the extracellular VFT module, thereby inducing the formation of a novel homodimer interface between subunits. (3) Ca2+ ions stabilize the active state by enhancing homodimer interactions between membrane-proximal domains to fully activate the receptor. The combination of agonist-induced VFT closure and specific association of membrane-proximal CR domains in CaSR will likely lead to rearrangement of the transmembrane domains for receptor activation.

Activation mechanism of CaSR.

Common class C GPCR activation mechanism

Structural and functional data suggest a universal activation mechanism for class C GPCRs. First, agonist causes VFT closure in all three receptor systems of CaSR, mGluRs and GABAB receptor. Second, receptor activation requires the association of membrane-proximal domains. For CaSR, this involves the formation of a novel homodimer interface between the LB2 and CR domains. For GABAB receptor, which lacks the CR region, agonist leads to the formation of a large heterodimer interface between the LB2 domains (Geng et al., 2013). Similarly for mGluRs, single-molecule fluorescence resonance energy transfer (FRET) studies indicate that the LB2 domains of mGluRs come into proximity to stabilize the active state (Vafabakhsh et al., 2015). Furthermore, disulfide crosslinking experiments of mGluR demonstrate that a precise association between the CR domains is sufficient for full receptor activation (Huang et al., 2011). Third, agonist binding is accompanied by a decrease in separation between the C-terminal ends of extracellular domains. This will likely result in rearrangement of the transmembrane domain dimer for receptor activation. Indeed, FRET and crosslinking studies have detected movement between interacting transmembrane domains of mGluRs and GABAB receptor upon activation (Matsushita et al., 2010; Tateyama et al., 2004; Xue et al., 2015). In conclusion, agonist-induced VFT closure that leads to the specific association of membrane-proximal domains is a common mechanism shared by all class C GPCRs during ligand-dependent receptor activation.

Materials and methods

Protein expression and purification

The extracellular domain of human CaSR (1–612) was cloned into the pFBDM vector (Berger et al., 2004) for expression in baculovirus-infected insect cells. Wild-type CaSR was heavily glycosylated, and we took two approaches to remove carbohydrates from CaSR ECD: (1) elimination of potential glycosylation sites through mutation, and (2) enzymatic deglycosylation.

Mutants of CaSR ECD were generated in which either two or three N-linked glycosylation sites were eliminated. Both mutant constructs contained the mutations N386Q and S402N, while one also had the additional mutation N468Q. A Flag tag was engineered at the C-terminus of each construct to facilitate affinity purification. In agreement with previous studies of full-length CaSR in mammalian cells (Ray et al., 1998), we found that expression of CaSR ECD in insect cells was essentially eliminated when more than three glycosylation sites were disrupted. The CaSR ECD glycosylation mutants were constructed to maximize the reduction of carbohydrate content while maintaining a sufficient expression level for structural studies.

Wild-type and mutant CaSR ECD were secreted from sf9 insect cells infected with the corresponding recombinant CaSR ECD virus. The CaSR ECD protein was isolated from cell supernatant by anti-Flag antibody (M2) affinity chromatography, and eluted with 100 µg/ml Flag peptide in 50 mM Tris, pH 7.5 and 150 mM NaCl. The protein was further purified by gel filtration chromatography (superdex 200, GE Healthcare Life Sciences, USA) in 20 mM Tris, pH 8.0 and 150 mM NaCl to remove aggregates. Finally, the CaSR ECD protein was applied to an ion exchange column (MonoQ, GE) in 20 mM Tris, pH 8.0 and eluted using a linear salt gradient from 0 to 1 M NaCl. All purification procedures were performed in the presence of 10 mM CaCl2. To isolate the L-Trp-bound receptor, purified CaSR ECD protein was washed extensively with a solution containing 20 mM Tris, pH 8.0, 10 mM L-Trp, and 10 mM CaCl2 prior to crystallization. This would facilitate the complete saturation of L-Trp- and Ca2+-binding sites within the receptor.

We applied an enzymatic deglycosylation procedure to wild-type receptor and the CaSR ECD mutant with two glycosylation-site mutations. Each construct was expressed in sf9 cells in the presence of the N-glycosylation processing inhibitor kifunensine (1 mg/L) to produce high mannose glycoproteins that were sensitive to enzymatic cleavage by endoglycosidase H (Endo H). The cell supernatant was applied to an M2 anti-Flag antibody affinity column, and the bound CaSR ECD protein was eluted with 100 µg/ml Flag peptide in 50 mM Tris, pH 7.5 and 150 mM NaCl. Well-folded CaSR ECD was separated from aggregates by gel filtration chromatography in 20 mM Tris, pH 8.0, and 150 mM NaCl. The CaSR ECD protein was subsequently digested overnight with Endo H in 50 mM Na Citrate, pH 5.5. The partially deglycosylated CaSR ECD protein was further purified by ion exchange chromatography in 20 mM Tris, pH8.0 using a linear salt gradient from 0 to 1 M NaCl. The protein purification and enzymatic deglycosylation procedures were performed either in the absence or presence of 2 mM CaCl2, which is within the normal range of Ca2+ concentrations in plasma.

Crystallization and data collection

Wild-type CaSR ECD did not form well-diffracting crystals possibly because the presence of flexible and heterogeneous carbohydrates on the protein surface interfered with crystallization.

The CaSR ECD mutant carrying two glycosylation-site mutations formed diffracting crystals after enzymatic deglycosylation. The crystals were obtained at 20°C in 1.5 M Li2SO4, 100 mM Tris pH 8.5 in the absence and presence of 2 mM CaCl2. Protein crystallization was achieved by hanging drop vapor diffusion method using 24-well VDX plates (Hampton Research, USA). The CaSR ECD crystals were flash-cooled with liquid nitrogen in a cryoprotecting solution containing 3.0 M Li2SO4, 100 mM Tris pH 8.5, and the same concentration of CaCl2 as used for crystallization. These CaSR ECD crystals have the space group F222 with one molecule per asymmetric unit (form I).

The CaSR ECD mutant carrying three glycosylation-site mutations crystallized under two different conditions. (1) In the absence of additional amino acids, CaSR ECD formed the best diffracting crystals at 20°C in 0.9 M Na Citrate, 100 mM Tris, pH 8.0, and 10 mM CaCl2. The crystals were obtained using hanging drop vapor diffusion technique. These crystals were flash-cooled with liquid nitrogen in a cryoprotecting solution containing 0.9 M Na Citrate, 100 mM Tris, pH 8.0, 10 mM CaCl2, and 20% glycerol. (2) Crystals of the L-Trp-bound CaSR ECD mutant were grown at 20°C in 1.6 M NaH2PO4, 0.4 M K2HPO4, 100 mM Na2HPO4/citric acid pH 4.2, 10 mM CaCl2, and 10 mM L-Trp. The crystals were obtained by sitting drop vapor diffusion method using 24-well Cryschem plates (Hampton Research). These crystals were flashed-cooled with liquid nitrogen in a cryoprotecting solution containing 20% glycerol and all other components of the crystallization solution. The crystals obtained from both conditions belong to space group C2 and have two molecules per asymmetric unit (form II).

Diffraction data were collected at the 24ID-C and 24ID-E beamlines of Advanced Photon Source (APS). To minimize radiation damage at low energy, a native dataset was collected at the energy of Se-K edge (λ=0.9792 Å) for a single form I crystal of CaSR ECD grown in the presence of 2 mM Ca2+. This native dataset was used to solve the inactive-state CaSR ECD structure. Form I crystals grown in the absence of additional Ca2+ diffracted to much lower resolution than those obtained in the presence of 2 mM Ca2+ and were therefore not used for structural determination.

Multiple anomalous data sets were also collected at λ = 1.7712 Å for form I crystals of CaSR ECD, each from a single crystal. A total of five anomalous data sets were obtained for CaSR ECD in the absence of Ca2+. These data sets were integrated individually by XDS (Kabsch, 2010), and then scaled and merged using CCP4 programs (Winn et al., 2011) to calculate the anomalous difference Fourier maps in the absence of additional Ca2+. Similarly, eight anomalous data sets were collected for CaSR ECD in the presence of 2 mM Ca2+, and combined to determine the anomalous difference Fourier maps in the presence of plasma concentration of Ca2+. The anomalous difference maps obtained in the absence and presence of Ca2+ revealed peaks that correspond to bound Ca2+ and SO42- ions at the same positions in the CaSR ECD structure. The averaging of multiple data sets enhanced signal to noise in anomalous diffraction (Liu et al., 2014).

A total of four anomalous data sets were collected at low energy (λ=1.7712 Å) for L-Trp-bound form II crystals of CaSR ECD, each from a single crystal. The four data sets were processed individually by XDS (Kabsch, 2010) and CCP4 programs (Winn et al., 2011). The data set with the highest resolution limit (2.6 Å) was used to determine the active-state CaSR ECD structure in the presence of L-Trp and excess Ca2+. The four data sets were also scaled and merged for the calculation of anomalous difference Fourier maps.

Structure determination

The structure of L-Trp-bound CaSR ECD in form II crystal was solved by molecular replacement. A two-fold non-crystallographic symmetry (NCS) axis was identified from the self-rotation function. Polyalanine models generated from the individual LB1 and LB2 domains of mGluR3 ECD structure (Muto et al., 2007) (PDB code: 2E4U) were used as search probes to locate the VFT modules of both CaSR ECD molecules in the crystal. After phase improvement by two-fold NCS-averaging, additional density appeared for the CR domain of CaSR ECD. A complete model of the CaSR ECD homodimer was developed through a succession of manual fittings and iterative refinement. The final model contained the CaSR ECD residues 20–119, 135–359 and 393–598 in one protomer, and residues 22–122, 136–360 and 392–602 in the other protomer. Each protomer contained eight intrasubunit disulfide bridges. Electron density was visible for carbohydrate residues (N-acetyl-glucosamine) attached to Asn90, Asn287, Asn488, Asn541 of one protomer, and Asn541 of its dimer partner. Finally, each protomer was also bound to one L-Trp ligand, four Ca2+ ions, and two PO43- ions. The Ca2+ and PO43- ions were identified by anomalous difference Fourier maps calculated using data collected at a wavelength of 1.7712 Å. We modeled PO43- as the anions in the CaSR ECD structure because they were major components of the crystallization solution. The anomalous scattering of Ca2+ at 1.75 Å has been used successfully to identify the Ca2+-binding sites in a voltage-gated calcium channel (Tang et al., 2014). Ramachandran analysis places 94.9% of all residues in favored regions and 0.28% in outlier regions.

A molecular replacement solution was also obtained for CaSR ECD in form II crystal in the presence of 10 mM Ca2+ but absence of any additional amino acid. The protein structure of L-Trp-bound CaSR ECD without any bound ligand was used as the search model. A stretch of electron density at the interdomain cleft region indicates the presence of an endogenous ligand. This structure was partially refined because we have not been able to identify the structure of the endogenous ligand by mass spectrometry despite multiple attempts.

The structure of partially deglycosylated CaSR ECD in form I crystal was solved using the individual LB1, LB2, and CR domains of L-Trp-bound CaSR ECD as search models. The asymmetric unit of the form II crystals contained one CaSR ECD protomer, and it forms a disulfide-linked homodimer with a crystallographic symmetry-related molecule. The final model for a single protomer contained CaSR ECD residues 21–130 and 136–603. In addition to the eight intrasubunit disulfide linkages within each protomer, the inter-subunit disulfide bond formed by C129 was ordered in the form I CaSR ECD crystal structure. Electron density was also visible for carbohydrate residues (N-acetyl-glucosamine) attached to Asn261, Asn287, Asn446, Asn468, Asn488, Asn541, and Asn594. In addition, each protomer had one Ca2+ ion, and three SO42- ions. The Ca2+ and SO42- ions were identified by anomalous difference Fourier maps calculated from data collected at 1.7712 Å. We reasoned that SO42- ions were most likely the anions bound to CaSR ECD since they were used as the precipitant for crystallization. Geometric analysis places 92.9% of all residues in favored regions and 0.35% as outliers. In the form I CaSR ECD structure, the interdomain groove was empty except for a water molecule; it also forms crystal contacts with a symmetry-related molecule.

Cell surface expression

Full-length human CaSR was cloned into a pcDNA3.1(+) vector (Life Technologies, USA) for expression in human embryonic kidney (HEK) 293 cells. A Flag tag was inserted after the signal peptide of CaSR. Mutants of CaSR were constructed using the QuikChange mutagenesis system (Agilent Technologies, California, USA).

HEK293 T/17 cells (ATCC) were transfected by Lipofectamine 2000 (Life Technologies) with wild-type or mutant CaSR plasmids. Cells permeabilized with 0.5% Triton X100 were used to determine the total expression level of CaSR in transfected cells. Untreated cells were used to determine the cell surface expression level of the receptor. The amount of surface protein detected for each construct was normalized to that found in the total cell lysate.

The cells were blocked with 5% milk, and then incubated with mouse anti-Flag M1 antibody (Sigma-Aldrich, USA) as the primary antibody to measure CaSR expression. Donkey anti-mouse IRDye 800-labeled antibody (LiCor Biosciences, Nebraska, USA) was used as the secondary antibody. Fluorescent signals were measured with an Odyssey Infrared Imager (LiCor). The results of three independent experiments were used for statistical analysis.

Most of the mutants were expressed on the cell surface at levels comparable to that of the wild-type receptor. The exceptions were R66H, R69E, I81M, T100I, N102I, and S417L, which reduced the surface expression of the mutant receptors to approximately 70–75% of the wild-type level.

Inositol phosphate measurement

Measurement of inositol phosphate (IP) accumulation was carried out using the homogenous time-resolved fluorescence (HTRF) IP-one Tb kit (Cisbio Bioassays, USA). This assay quantifies the accumulation of inositol 1-monophosphate (IP1), a degradation product of inositol 1,4,5-triphosphate (IP3) that is stable in the presence of LiCl. Briefly, HEK293 T/17 cells were transiently transfected with wild-type or mutant full-length CaSR plasmids. The cells were stimulated with increasing concentrations of Ca2+ two days post transfection in a buffer containing 10 mM HEPES pH 7.4, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, and 50 mM LiCl. The reaction mixture was then incubated with an IP1 analog coupled to a d2 fluorophore (acceptor) and an anti-IP1 monoclonal antibody labeled with Eu Cryptate (donor). The IP1 produced by cells upon activation of CaSR competes with IP1 coupled to the dye d2 for binding to the anti-IP1 antibody. The resulting FRET signal is inversely proportional to the concentration of IP1 in the sample. The fluorescence data was acquired at 620 and 665 nm using an EnVision plate reader (Perkin Elmer, USA) after laser excitation at 320 nm. The FRET signal was calculated as the fluorescence ratio (665 nm/620 nm). Basal activity was determined in the absence of Ca2+ stimulation. The percent stimulation of each receptor mutant was calculated based on the wild-type response obtained under the same condition. Data analysis was performed using the non-linear regression algorithms in Prism (GraphPad Software, USA). Data points represent average ± s.e.m. of triplicate measurements.

The effect of anion on Ca2+-stimulated IP accumulation was determined using SO42- instead of PO43- because of the modest solubility of CaHPO4 (1.5 mM), the predominant form of calcium phosphate salt at physiological pH (7.4). Specifically, dose-dependent Ca2+-induced IP accumulation was measured in the absence and presence of 10 mM Li2SO4 in the reaction buffer.

Binding of 0.1 mM L-[3H]Trp (1 Ci/mmol) to 600 ng of purified CaSR ECD was measured with the scintillation proximity assay (SPA) using 1.25 mg/mL Yttrium silicate (YSi) protein A SPA beads (PerkinElmer, USA) in conjunction with 0.125 ng/mL of anti-Flag M2 antibody in 20 mM HEPES, pH 8.0 and 150 mM NaCl at 4°C. Increasing concentrations of non-radioactive L-Trp were added to compete for receptor binding in the presence of 0 mM or 2 mM CaCl2. Each reaction was also performed in the presence of 60 mM non-radioactive L-Trp for background correction. The reactions were allowed to proceed for 1 hr to reach equilibrium. The plates were then counted in a Microbeta counter (PerkinElmer, USA). Data were analyzed using the non-linear regression algorithms in Prism (GraphPad).

Decision letter

Ehud Y Isacoff

Reviewing Editor; University of California, Berkeley, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Structural mechanism of ligand activation in human calcium-sensing receptor" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Ehud Isacoff as a guest Reviewing Editor and Randy Schekman as the Senior Editor. One of the three reviewers has agreed to reveal their identity: Yoshihiro Kubo.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Geng et al. describe the first crystal structures of the calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR). These structures of the extracellular domain demonstrate that L-Trp is an orthosteric agonist of the receptor and reveal binding sites for modulating Ca2+ and PO43- ions. Structures in four pharmacologically relevant conditions reveal two distinct conformations consistent with a model of an activation rearrangement that is triggered by venus flytrap closure on agonist, which positions the cysteine-rich domains of the subunits in close proximity – consistent with earlier structural and biochemical analysis on other class C GPCRs. The study suggests that positive and negative allosteric modulation by Ca2+ and PO43- ions act occurs via modulation of the binding of the orthosteric agonist.

Essential revisions:

1) Evaluate the L-Trp EC50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).

2) To demonstrate the inhibitory effect of PO43- on the response of CaSR to Ca2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites.

3) To demonstrate the potentiating effect of L-Trp on the response to Ca2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-Trp, in wt and mutants of the L-tryptophan binding sites.

Reviewer #1:

The manuscript by Geng et al. describes the crystal structures of the human calcium-sensing G protein-coupled receptor extracellular domain (ECD). In a nutshell, these structures demonstrate that L-Trp is an orthosteric agonist of the receptor and reveal several binding sites for Ca2+ and PO43- ions that, based on mutagenesis data and functional assays, may play a key role in balancing receptor activity.

In particular, the structures were obtained in four different pharmacologically relevant conditions (with or without Ca2+ and L-Trp) and revealed only two distinct conformations. Based on the structural information available for the same receptor family (class C GPCRs), the authors propose that these two conformations represent inactive and active states of the receptor. They provide a detailed comparison of both structures to highlight the rearrangement of the ECDs that is necessary for receptor activation. Of note, this is the first structure in the class C family in which the cystein rich domain (CRD) adopt such a conformation in an agonist bound condition. This structure nicely fits the crosslinking data obtained for the metabotropic glutamate receptors in living cells and thus validate the ECD activation model of class C GPCR possessing a CRD domain: an interdomain movement triggered by VFT closure which positions the CRDs in close proximity.

I thus believe this study is of immediate interest to many people in the GPCR field and beyond. Overall the methodology and data are robust and I did not find any flaws that should prohibit its publication.

To conclude, I think it is tempting to speculate that the Ca2+ and PO43- ions act respectively as positive and negative allosteric modulators of the CaSR receptor activity by modulating, at least in part, the binding of the orthosteric agonist L-Trp. One key experiment would thus be to evaluate the L-Trp EC50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).

Although this might strengthen the manuscript, I do not believe these data would be critical for publication.

Reviewer #2:

This is an important manuscript reporting the first crystal structure of CaSR. The structure showed interesting features in terms of ligand binding sites. The binding site for Ca2+, a main agonist, was different from the canonical ligand binding sites identified in other class C GPCRs, such as mGluR and GABABR. It is noteworthy that this canonical site in CaSR was occupied by L-tryptophan, a putative co-agonist. These features concerning about binding sites and the proposed activation mechanism of CaSR is unique, in comparison to other class C GPCRs. Therefore, this paper will contribute to facilitate the understanding of physiological and biophysical properties of CaSR and class C GPCR. The authors also identified the putative binding sites for PO43-, and suggested that PO43- is an antagonist of CaSR for the first time.

I highly evaluate the scientific merits and the general impact of the paper. I judge it is worthy for publication, but only after satisfactory revisions. I have four major comments which require attention.

1) Experiments to demonstrate the inhibitory effect of PO43-

The conclusion in the Discussion that " PO43- ions promote the inactive configuration" is rather speculative from the two reasons in the following.

A) There are no direct evidence which shows PO43- promotes the inactivated conformation. The authors showed mutants of the PO43- binding sites have lower IP accumulation in response to extracellular Ca2+. I think the data are not sufficient. To demonstrate the inhibitory effect of PO43- on the response of CaSR to extracellular Ca2+ conclusively, I would like to request to present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites. The expected result is lower response to Ca2+ in the presence of PO43- than in the absence, not in mutants but in wt.

B) The authors assume that the electron density represents the presence of PO43- or SO43-, because there are no anions in the crystallization solution other than PO43- or SO43-. Is there a possibility that carryover endogenous anion(s) other than PO43- is bound to the anion binding site? (similarly to the case of the putative carryover of endogenous amino acid(s) at the L-tryptophan binding site when crystalized in the absence of L-tryptophan).

2) Experiments to demonstrate the potentiating effect of L-tryptophan

I have similar comments to the case of anion effect. To demonstrate the potentiating effect of L-tryptophan on the response of CaSR to extracellular Ca2+ conclusively, please present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-tryptophan, in wt and mutants of the L-tryptophan binding sites. The expected result is higher response to Ca2+ in the presence of L-tryptophan than in the absence, not in mutants but in wt.

3) Comparison with the mGluR and GABABR

The authors discussed differences and similarities of the CaSR structure to those of mGluR and GABABR. However, it is hard to follow the explanations in the text, without structure images for comparison.

A) The authors mentioned the degree of rotational change of two LB1 interface in CaSR with GABAB and mGluR upon activation (subsection “Common protomer-protomer interactions”, third paragraph). In Figure 3—figure supplement 1B, D, the authors showed two images of CaSR in the resting and activated states. I would like to request to add similar images in the two states of mGluR and GABABR in Figure 3—figure supplement 1, to clarify the discussion of the rotated angle of LB1 interface.

B) The authors described an overall similarity of VFT cleft structure of CaSR and mGluR. They also wrote the tryptophan binding site of CaSR corresponds to the glutamate binding site of mGluR (subsection “Structure of CaSR ECD homodimer”, second paragraph). To clarify these points, please present a superimposed image of the VFT cleft of CaSR and mGluR, in e.g. Figure 1—figure supplement 1.

4) Gd3+ binding site

In the crystal structure of mGluR1, a binding site for Gd3+ was identified at the dimer interface (Tsuchiya et al., PNAS 2002). I would like to have explanations whether or not a similar structure was identified in CaSR.

Reviewer #3:

Geng et al. reported the crystal structures of entire extracellular domain of calcium sensing receptor of calcium free form and loaded forms in the presence of calcium and PO43- ions. This work is of importance for providing insights for the activation mechanism via dimer interface between subunits. There are several major concerns that need be addressed:

1) The crystal structures were determined for CaSR with mutations of either two (N386Q and S402N) or three (N468Q) N-glycosylation sites. However, no study has been carried out on the effects of these mutations on expression and function of CaSR. It is important to provide evidence with experimental data that mutations at these glycosylation sites do not result in alteration of the function and expression.

2) Since mutations could lead to reduction in cell surface expression due to trafficking, it is encouraged to provide confocal images to ensure that the observed change in intracellular IP activity is not due to reduction of protein expression levels on cell surface.

3) The identification of Trp binding site is very important for the activation mechanism. It will be very helpful to report Trp binding affinity of the ECD of CaSR.

4) Extremely high concentrations of three different anion ions were used for crystallization. The role of anion binding is not clear and some of them appear to be non-specific. Among 4 anion-binding sites identified, sites 1-3 are in inactive form crystalized with 3.0 M Li2SO4, 100 mM Tris pH8.5 with mutations N386Q and S402N. Under this condition, SO42- ions are assigned. On the other hand, 1.6 M NaH2PO4, 0.4 M K2HPO4, 100 mM Na2HPO4/citric acid pH4.2, 10 mM Trp 10 mM Ca2+ are used for identification of anion sites in the active form of CaSR with three glycosylation mutations. Thus, 2 anion binding sites were modeled to be PO43. What is the binding affinity for these anion ions?

5) The four calcium binding sites shown in Figure 5 either have incomplete coordination or lack of negatively charged residues. These properties are very different from classical calcium binding sites with at least three ligand oxygen atoms from the protein and with electrostatic interactions. The reported site 3 calcium is directly chelated only by one oxygen from hydroxyl side chin of S302. Site 4 is formed by side chain of D234 and mainchain carbonyl oxygen from E231 and G557. How the function results provided in Figure 6 is a mutation G55E that results in a significantly increase of EC50 for IP accumulation and intracellular calcium response. Why have such big alteration of activity for a replacement of another carbonyl oxygen? Since calcium interaction is largely electrostatic, it would make sense to test the mutational effect D234A. The provided mutational studies of I181 m, T100I, and N102I all have significantly reduced maximal amplitudes in both IP and intracellular calcium activities in addition to increased EC50. These results suggest that the surface expression of the mutated protein variants are largely reduced. Thus, the effect for these residues in calcium binding cannot be unambiguously revealed. It is important to provide the results for the mutational effect of these identified ligand residues on direct calcium binding affinity.

6) It is worth to pointing out that at pH4.2 sidechain of Asp and Glu are largely protonated with significantly reduced calcium binding capability. In addition, due to poor solubility of calcium phosphate, the molar phosphate concentrations used in the buffer will result in extremely low concentration of soluble free calcium. Thus, the role of calcium binding in activation revealed in the determined structures is likely to be under estimated due to the condition used for crystallization.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Structural mechanism of ligand activation in human calcium-sensing receptor" for further consideration at eLife. Your revised article has been favorably evaluated by Randy Schekman (Senior Editor), a Reviewing Editor, and one reviewer.

The manuscript has been improved but there is a remaining issue that needs to be addressed before acceptance, as outlined below:

1) In the point by point response to Reviewer #3, comment #3, the authors stated the following:

"The presence of 2 mM Ca2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR."

The authors should clarify what they mean by this. If calcium stabilized the Trp-bound state it would presumably be seen as an increase in Trp affinity. An increase in amount of radio-Trp that is bound that does not shift the affinity curve would seem to suggest adding to circulation Trp binding sites (i.e. receptors) that were previously not available (from an unavailable/inactive pool?). Or is there another possible explanation? Or does this raise a caveat about the experiment? This should be addressed in the text.

Reviewer #1:

I believe the revised version of the manuscript "Structural mechanism of ligand activation in human calcium-sensing receptor" is addressing the essential revisions that were raised during the first reviewing process. In particular, the authors have addressed the allosteric effect of Ca2+ on L-Trp activity (and vice versa).

Author response

Essential revisions:

1) Evaluate the L-Trp EC50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).

Following the referees’ suggestion, we have evaluated the L-Trp EC50 at different Ca2+ concentrations by monitoring changes in CaSR-mediated intracellular Ca2+ response in single cells. In agreement with previous findings, L-Trp can activate CaSR when the extracellular Ca2+ concentration is above the threshold level of 0.5 mM. The effect of L-Trp on CaSR was concentration-dependent, with an apparent EC50 of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR. The new data are presented in Figure 4E and 4F.

2) To demonstrate the inhibitory effect of PO43- on the response of CaSR to Ca2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites.

Following the referees’ suggestion, we have measured the response of CaSR to extracellular Ca2+ in the absence and presence of the anion SO42-. We chose SO42- to substitute for PO43- because CaSO4 is more soluble than CaHPO4, the predominant form of calcium phosphate salt at physiological pH. At pH 7.4, approximately 61.3% of the phosphate ions exist in the form of HPO42- and 38.7% are in the form of H2PO4-. The solubility of CaHPO4 and Ca(H2PO4)2 are 1.5 mM and 71 mM, respectively. The solubility of CaSO4 is 15 mM.

As predicted by the referee, and consistent with our structural observation, we found that the response of CaSR to Ca2+ was slightly lower in the presence of than in the absence SO42-. This confirms that anions have a negative allosteric effect on the receptor. The new data are presented in Figure 8C.

We have also examined the effects of SO42- on mutants of the anion binding-sites. Our structures revealed that the anion-binding residues R62 at site 1 and R66 at site 3 form hydrogen bonds across the interdomain cleft in the absence of bound anions to stabilize the closed conformation of the receptor. We found that the effect of SO42- on Ca2+-induced activation of the R62M mutant was similar to that of the wild-type receptor, while the presence of SO42- had little effect on the activity of the R66H mutant. However, we cannot conclude which anion-binding sites are involved in mediating the inhibitory effect of anions because these mutations are expected to have both positive and negative effects on the receptor function.

A) The mutations R62M and R66H are expected to eliminate the inhibitory effect of anions by preventing their binding to the receptor.

B) These same mutations would also disrupt the formation of hydrogen bonds across the interdomain cleft by R62 and R66, thereby eliminating the interactions that favor receptor activation.

C) Residue R66 is involved in the binding of anion at both sites 2 and 3. Since anion-binding site 2 appears to be crucial for structural integrity of the receptor, disruption of this site by the R66H mutation would negatively affect receptor function.

3) To demonstrate the potentiating effect of L-Trp on the response to Ca2+, compare responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-Trp, in wt and mutants of the L-tryptophan binding sites.

Following the referee’s suggestion, we have measured the potentiating effect of L-Trp on the response of wild-type CaSR to extracellular Ca2+. During the course of our study, we found that it was very difficult to measure the functional effect of L-Trp through IP accumulation. Instead, we monitored the effect of L-Trp on CaSR-mediated intracellular Ca2+ mobilization. As predicted by the referee, Ca2+-induced receptor activation is higher in the presence of L-Trp than in the absence. The new data are presented in Figure 4G.

We have shown using IP accumulation measurements that most alanine mutants of the L-Trp- binding site abolished Ca2+-dependent receptor response in the absence of any amino acid. Therefore, these mutants would not be suitable for the study of the L-Trp effect. For the two mutants that displayed partial receptor activity (S147A and Y218S), we found that they did not respond to 10 mM L-Trp in the presence of 2 mM extracellular Ca2+.

Effect of various concentrations of L-Trp (1, 2, 0, 10 mM) on intracellular Ca2+ mobilization in the presence of 2 mM extracellular Ca2+.

I think it is tempting to speculate that the Ca2+ and PO43- ions act respectively as positive and negative allosteric modulators of the CaSR receptor activity by modulating, at least in part, the binding of the orthosteric agonist L-Trp. One key experiment would thus be to evaluate the L-Trp EC50 in cell-based assays using different ionic conditions (such as no Ca2+ to 10 mM Ca2+).

Although this might strengthen the manuscript, I do not believe these data would be critical for publication.

Following the referee’s suggestion, we have evaluated the L-Trp EC50 at different Ca2+ concentrations by monitoring changes in CaSR-mediated intracellular Ca2+ response in single cells. In agreement with previous findings, L-Trp can activate CaSR when the extracellular Ca2+ concentration is above the threshold level of 0.5 mM. The effect of L-Trp on CaSR was concentration-dependent, with an apparent EC50 of 0.12 ± 0.06 mM when extracellular Ca2+ was present at 2.5 mM. The efficacy of L-Trp decreased at lower concentrations of Ca2+ that are within the physiological range (L-Trp EC50 = 0.75 ± 0.51 mM at 1.5 mM Ca2+), consistent with previous proposal that multiple amino acids need to act in concert to control the function of CaSR. The new data are presented in Figure 4E and 4F.

Reviewer #2:

I highly evaluate the scientific merits and the general impact of the paper. I judge it is worthy for publication, but only after satisfactory revisions. I have four major comments which require attention.

1) Experiments to demonstrate the inhibitory effect of PO43-

The conclusion in the Discussion that " PO43- ions promote the inactive configuration" is rather speculative from the two reasons in the following.

A) There are no direct evidence which shows PO43- promotes the inactivated conformation. The authors showed mutants of the PO43- binding sites have lower IP accumulation in response to extracellular Ca2+. I think the data are not sufficient. To demonstrate the inhibitory effect of PO43- on the response of CaSR to extracellular Ca2+ conclusively, I would like to request to present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of PO43-, in wt and mutants of the anion binding sites. The expected result is lower response to Ca2+ in the presence of PO43- than in the absence, not in mutants but in wt.

Following the referee’s suggestion, we have measured the response of CaSR to extracellular Ca2+ in the absence and presence of the anion SO42-. We chose SO42- to substitute for PO43- because CaSO4 is more soluble than CaHPO4, the predominant form of calcium phosphate salt at physiological pH. At pH 7.4, approximately 61.3% of the phosphate ions exist in the form of HPO42- and 38.7% are in the form of H2PO4-. The solubility of CaHPO4 and Ca(H2PO4)2 are 1.5 mM and 71 mM, respectively. The solubility of CaSO4 is 15 mM.

As predicted by the referee, and consistent with our structural observation, we found that the response of CaSR to Ca2+ was slightly lower in the presence of than in the absence SO42-. This confirms that anions have a negative allosteric effect on the receptor. The new data are presented in Figure 8C.

We have also examined the effects of SO42- on mutants of the anion binding-sites. Our structures revealed that the anion-binding residues R62 at site 1 and R66 at site 3 form hydrogen bonds across the interdomain cleft in the absence of bound anions to stabilize the closed conformation of the receptor. We found that the effect of SO42- on Ca2+-induced activation of the R62M mutant was similar to that of the wild-type receptor, while the presence of SO42- had little effect on the activity of the R66H mutant. However, we cannot conclude which anion-binding sites are involved in mediating the inhibitory effect of anions because these mutations are expected to have both positive and negative effects on the receptor function.

A) The mutations R62M and R66H are expected to eliminate the inhibitory effect of anions by preventing their binding to the receptor.

B) These same mutations would also disrupt the formation of hydrogen bonds across the interdomain cleft by R62 and R66, thereby eliminating the interactions that favor receptor activation.

Residue R66 is involved in the binding of anion at both sites 2 and 3. Since anion-binding site 2 appears to be crucial for structural integrity of the receptor, disruption of this site by the R66H mutation would negatively affect receptor function.

B) The authors assume that the electron density represents the presence of PO43- or SO43-, because there are no anions in the crystallization solution other than PO43- or SO43-. Is there a possibility that carryover endogenous anion(s) other than PO43- is bound to the anion binding site? (similarly to the case of the putative carryover of endogenous amino acid(s) at the L-tryptophan binding site when crystalized in the absence of L-tryptophan).

We agree with the referee that it is possible carryover endogenous anion(s) other than PO43- or SO42- is bound at the anion-binding sites. We have added a sentence in the text to mention the caveat.

2) Experiments to demonstrate the potentiating effect of L-tryptophan

I have similar comments to the case of anion effect. To demonstrate the potentiating effect of L-tryptophan on the response of CaSR to extracellular Ca2+ conclusively, please present data comparing the responses (IP accumulation) to extracellular Ca2+ in the presence and absence of L-tryptophan, in wt and mutants of the L-tryptophan binding sites. The expected result is higher response to Ca2+ in the presence of L-tryptophan than in the absence, not in mutants but in wt.

Following the referee’s suggestion, we have measured the potentiating effect of L-Trp on the response of wild-type CaSR to extracellular Ca2+. As predicted by the referee, Ca2+-induced receptor activation is higher in the presence of L-Trp than in the absence. The new data are presented in Figure 4G.

We have shown using IP accumulation measurements that most alanine mutants of the L-Trp- binding site abolished Ca2+-dependent receptor response in the absence of any amino acid. Therefore, these mutants would not be suitable for the study of the L-Trp effect. For the two mutants that displayed partial receptor activity (S147A and Y218S), we found that they did not respond to 10 mM L-Trp in the presence of 2 mM extracellular Ca2+.

3) Comparison with the mGluR and GABABR

The authors discussed differences and similarities of the CaSR structure to those of mGluR and GABABR. However, it is hard to follow the explanations in the text, without structure images for comparison.

A) The authors mentioned the degree of rotational change of two LB1 interface in CaSR with GABAB and mGluR upon activation (subsection “Common protomer-protomer interactions”, third paragraph). In Figure 3—figure supplement 1 B, D, the authors showed two images of CaSR in the resting and activated states. I would like to request to add similar images in the two states of mGluR and GABABR in Figure 3—figure supplement 1, to clarify the discussion of the rotated angle of LB1 interface.

Following the referee’s recommendation, we have added images of the two states of mGluR1 and GABAB receptor comparing the rotated angle of the LB1-LB1 interface in each receptor system (Figure 3—figure supplement 1).

B) The authors described an overall similarity of VFT cleft structure of CaSR and mGluR. They also wrote the tryptophan binding site of CaSR corresponds to the glutamate binding site of mGluR (subsection “Structure of CaSR ECD homodimer”, second paragraph). To clarify these points, please present a superimposed image of the VFT cleft of CaSR and mGluR, in e.g. Figure 1—figure supplement 1.

We agree with the referee and have added a superimposed image of the agonist-binding cleft of CaSR and mGluR1 in Figure 4—figure supplement 1.

4) Gd3+ binding site

In the crystal structure of mGluR1, a binding site for Gd3+ was identified at the dimer interface (Tsuchiya et al., PNAS 2002). I would like to have explanations whether or not a similar structure was identified in CaSR.

We think the referee’s question is very interesting, and have superimposed the structures of CaSR and mGluR to compare this region of the dimer interface. As shown in Author response image 3, the Gd3+ ion in the mGluR1 structure is coordinated by two acid residues, E238 and D242 from each subunit. The corresponding residues in the CaSR structure are R220 and E224. The basic residue R220 would not be compatible with the binding of a cation to CaSR at this location.

Superposition of the CaSR and mGluR1 structures in the region of the Gd3+-binding site in mGluR1 structure.

Geng et al. reported the crystal structures of entire extracellular domain of calcium sensing receptor of calcium free form and loaded forms in the presence of calcium and PO43- ions. This work is of importance for providing insights for the activation mechanism via dimer interface between subunits. There are several major concerns that need be addressed:

1) The crystal structures were determined for CaSR with mutations of either two (N386Q and S402N) or three (N468Q) N-glycosylation sites. However, no study has been carried out on the effects of these mutations on expression and function of CaSR. It is important to provide evidence with experimental data that mutations at these glycosylation sites do not result in alteration of the function and expression.

We agree with the referee that it is important to provide evidence that the glycosylation mutations introduced into our expression constructs do not alter the function of the receptor. It has been reported previously that glycosylation is not required for signal transduction of CaSR (Ray et al., JBC, 1998, 273, 34558-34567). Following the referee’s suggestion, we have also generated two full-length CaSR constructs for functional analysis in mammalian cells, one carrying the two glycosylation-site mutations N386Q and S402N, and the other carrying the additional mutation N468Q. We found that Ca2+-induced IP accumulation was statistically the same for the wild-type and mutant receptors, indicating that these glycosylation mutations did not alter CaSR function (Figure 1—figure supplement 1).

On the other hand, previous studies of full-length CaSR in mammalian cells indicate that disruption of the glycosylation sites impairs proper processing and expression of the receptor at the cell surface (Ray et al., JBC, 1998, 273, 34558-34567). In agreement with these findings, we found that expression of CaSR ECD in insect cells was essentially eliminated when more than three glycosylation sites were mutated. After testing mutations at each of the potential N-linked glycosylation sites individually and in various combinations, we chose the constructs reported in this study to maximize the reduction of carbohydrate content while maintaining a sufficient expression level for structural analyses.

2) Since mutations could lead to reduction in cell surface expression due to trafficking, it is encouraged to provide confocal images to ensure that the observed change in intracellular IP activity is not due to reduction of protein expression levels on cell surface.

We appreciate the referee’s suggestion. Unfortunately, we do not have any experience with confocal imaging. We did measure the cell surface expression levels of wild-type and mutant CaSR by on-cell western analysis, which was described in the Methods section. Most of the mutants in our study were expressed on the cell surface at levels comparable to that of the wild- type receptor. The exceptions were R66H, R69E, I81M, T100I, N102I, and S417L, which reduced the surface expression of the mutant receptors to approximately 70-75% of the wild-type level. Nevertheless, the reduction in the protein expression level on cell surface could not account for the much greater decrease in intracellular IP accumulation for each of these mutants. We therefore concluded that the mutations affected receptor function.

3) The identification of Trp binding site is very important for the activation mechanism. It will be very helpful to report Trp binding affinity of the ECD of CaSR.

We agree with the referee, and have determined the binding affinity of L-Trp for CaSR ECD by scintillation proximity assay using radiolabeled [3H]-Trp. In the absence of Ca2+, the binding of [3H]-L-Trp to CaSR ECD was inhibited by nonradioactive L-Trp with a half-maximal inhibitory concentration (IC50) of 2.1 ± 0.7 mM. The presence of 2 mM Ca2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR.

4) Extremely high concentrations of three different anion ions were used for crystallization. The role of anion binding is not clear and some of them appear to be non-specific. Among 4 anion-binding sites identified, sites 1-3 are in inactive form crystalized with 3.0 M Li2SO4, 100 mM Tris pH8.5 with mutations N386Q and S402N. Under this condition, SO42- ions are assigned. On the other hand, 1.6 M NaH2PO4, 0.4 M K2HPO4, 100 mM Na2HPO4/citric acid pH4.2, 10 mM Trp 10 mM Ca2+ are used for identification of anion sites in the active form of CaSR with three glycosylation mutations. Thus, 2 anion binding sites were modeled to be PO43-. What is the binding affinity for these anion ions?

We agree with the referee that it would be informative to determine the binding affinities of the anions to CaSR. We tried to quantify the binding of PO43- ions to purified CaSR ECD protein using microscale thermophoresis, but were unsuccessful. Several factors may have contributed to the difficulties we encountered. (1) The anions at different sites may have overlapping binding affinity ranges that cannot be easily differentiated. (2) Some sites such as site 2 may already be occupied by endogenous anions. (3) Low-affinity anion-receptor interactions at some sites are difficult to detect.

5) The four calcium binding sites shown in Figure 5 either have incomplete coordination or lack of negatively charged residues. These properties are very different from classical calcium binding sites with at least three ligand oxygen atoms from the protein and with electrostatic interactions. The reported site 3 calcium is directly chelated only by one oxygen from hydroxyl side chin of S302. Site 4 is formed by side chain of D234 and mainchain carbonyl oxygen from E231 and G557. How the function results provided in Figure 6 is a mutation G55E that results in a significantly increase of EC50 for IP accumulation and intracellular calcium response. Why have such big alteration of activity for a replacement of another carbonyl oxygen? Since calcium interaction is largely electrostatic, it would make sense to test the mutational effect D234A.

We appreciate the referee’s observation that the coordination of Ca2+ ions at all four binding sites in CaSR appear different from classical Ca2+-binding sites. It is possible that water molecules play a role in coordinating the bound Ca2+ ions, but could not all be seen in the structures due to disordering or the modest resolution of the structures.

The alteration in receptor activity of the G557E mutant may have resulted from a change in the conformation of the loop containing residue 557 such that the carbonyl oxygen is no longer optimally positioned to coordinate the binding of a Ca2+ ion at this site. Nevertheless, the effect of the G557E mutation is not as substantial as some of the other Ca2+-binding site mutations including I81M, T100I and N102I, which nearly abolished Ca2+-induced receptor activation. The EC50 for Ca2+-induced IP accumulation increased approximately two fold from 1.6 mM for the wild-type receptor to 3.4 mM for the G557E mutant, while the Emax of the receptor response remained essentially the same.

Following the referee’s suggestion, we have tested the mutation D234A, and found that its effect on receptor activity was negligible, as shown by the IP accumulation dose response curves for wild-type receptor and the D234A mutant (Author response image 4). This may be due to the fact that the side chain of D234 is somewhat flexible, and does not contribute to the binding of Ca2+ ion at site 4 as much as the backbone carbonyl oxygen atoms of E231 and G557.

The provided mutational studies of I181 m, T100I, and N102I all have significantly reduced maximal amplitudes in both IP and intracellular calcium activities in addition to increased EC50. These results suggest that the surface expression of the mutated protein variants are largely reduced. Thus, the effect for these residues in calcium binding cannot be unambiguously revealed. It is important to provide the results for the mutational effect of these identified ligand residues on direct calcium binding affinity.

The cell surface expression levels of the mutants I81M, T100I, N102I were approximately 70- 75% of the wild-type level based on on-cell western measurements. However, each of these mutations nearly abolished Ca2+-dependent receptor response, indicating that their effects on calcium binding and receptor function could not be explained by the reduction in cell surface expression alone.

We agree with the referee that it would be very informative to measure the direct binding affinity of Ca2+ at each site, and the mutational effects of Ca2+-binding residues. We tried two different approaches to determine the Ca2+-binding affinity to wild-type CaSR ECD, including microscale thermophoresis and scintillation proximity assay. Unfortunately, we were not successful possibly for the same reasons that we had difficulties measuring the affinities of anions. (1) The Ca2+ ions at different sites may have overlapping binding affinity ranges that cannot be easily differentiated. (2) Some sites such as site 2 may already be occupied by endogenous Ca2+. (3) Low-affinity Ca2+-receptor interactions at some sites are difficult to detect.

6) It is worth to pointing out that at pH4.2 sidechain of Asp and Glu are largely protonated with significantly reduced calcium binding capability. In addition, due to poor solubility of calcium phosphate, the molar phosphate concentrations used in the buffer will result in extremely low concentration of soluble free calcium. Thus, the role of calcium binding in activation revealed in the determined structures is likely to be under estimated due to the condition used for crystallization.

We think the referee raised an important question. However, we do not think that the role of calcium binding in activation as revealed by the determined structures is likely to be underestimated for two reasons.

1) Based on the following calculation, the proton concentration at pH4.2 is approximately 0.1 mM, much lower than the concentration of free Ca2+ (10 mM). Therefore, we think it is more likely for the sidechains of Asp and Glu residues to be bound to Ca2+ ion than protonated.

pH = -log[H+] = 4.2

[H+] = 1 x 10-4.2 M ≈ 0.1 mM

2) We think the concentration of soluble free calcium under the pH 4.2 crystallization condition should remain at 10 mM because most of the phosphate ions are in the form of H2PO4-, and the solubility of Ca(H2PO4)2 is approximately 71 mM.

Three equilibrium reactions exist between H3PO4, H2PO4-, HPO42-, and PO43-

Equilibrium

Constant

Value

Equilibrium

Ka1

7.5 x 10-3

H3PO4 <--> H+ + H2PO4-

Ka2

6.2 x 10-8

H2PO4- <--> H+ + HPO42-

Ka3

4.8 x 10-13

HPO42- <--> H+ + PO43-

H3PO4 <--> H+ + H2PO4-

pKa1 = pH - log([H2PO4-]/[H3PO4])

-log(7.5 x 10-3) = 4.2 - log([H2PO4-]/[H3PO4])

[H2PO4-]/[H3PO4] = 102.1

H2PO4- <--> H+ + HPO42-

pKa2 = pH - log([HPO42-]/[H2PO4-])

-log(6.2 x 10-8) = 4.2 - log([HPO42-]/[H2PO4-])

[H2PO4-]/[HPO42-] = 103

HPO42- <--> H+ + PO43-

pKa3 = pH - log([PO43-]/[HPO42-])

-log(4.8 x 10-13) = 4.2 - log([PO43-]/[HPO42-])

[HPO42-]/[PO43-] = 108.1

[H2PO4-]/[PO43-] = 1011.1

Therefore, more than 99% of the phosphate ions exist in the form of H2PO4- at pH4.2.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Structural mechanism of ligand activation in human calcium-sensing receptor" for further consideration at eLife. Your revised article has been favorably evaluated by Randy Schekman (Senior Editor), a Reviewing Editor, and one reviewer.

The manuscript has been improved but there is a remaining issue that needs to be addressed before acceptance, as outlined below:

1) In the point by point response to Reviewer #3, comment #3, the authors stated the following:

"The presence of 2 mM Ca2+ did not affect the binding affinity of L-Trp, indicating that Ca2+ ion is not directly involved in L-Trp recognition. This is consistent with our structural observation that no Ca2+ ion is found at the L-Trp-binding site. Nevertheless, Ca2+ ion increased the amount of L-Trp bound to CaSR ECD at any given concentration, indicating that extracellular Ca2+ enhances the L-Trp-binding site occupancy, possibly by stabilizing the L-Trp-bound and active conformation of CaSR."

The authors should clarify what they mean by this. If calcium stabilized the Trp-bound state it would presumably be seen as an increase in Trp affinity. An increase in amount of radio-Trp that is bound that does not shift the affinity curve would seem to suggest adding to circulation Trp binding sites (i.e. receptors) that were previously not available (from an unavailable/inactive pool?). Or is there another possible explanation? Or does this raise a caveat about the experiment? This should be addressed in the text.

We think referee #3 has raised a valid criticism. As pointed out by referee #1 below, we cannot conclude based on our [3H]-L-Trp binding competition studies alone that the binding affinity of L-Trp is the same with or without Ca2+. Given our structural observation that Ca2+ stabilizes the active conformation of the receptor, we agree that Ca2+ would most likely enhance the binding affinity of L-Trp since the interaction of L-Trp with an active receptor is expected to be more extensive than with an inactive receptor. In an active receptor, L-Trp would contact both the LB1 and LB2 domains. L-Trp may still bind to the open cleft in an inactive receptor, but its interaction with the receptor will be limited to the LB1 domain, which would result in lower affinity. The binding of glutamate to an open cleft has previously been observed for mGluR1 (Kunishima et al., Nature 2000, 407, 971-977).

Unfortunately, as indicated by referee #1, it would be difficult for us to measure the binding affinity of L-Trp in a saturation binding experiment since the concentration of [3H]-L-Trp required would be ≥10 mM, and a relatively low specific radioactivity of the commercially available radio-labeled ligand may preclude such experiments. Our competition experiments in the current study were performed at 0.1 mM [3H]-L-Trp, a concentration that is about 20-fold below the determined half-maximal inhibitory concentration (IC50) of non-labeled L-Trp for replacing radiolabeled L-Trp.

In addition, the presence of Ca2+ may affect the on- and off-rate of L-Trp binding to CaSR ECD. Specifically, L-Trp may have a slower off-rate when bound to the active conformation. Therefore, the higher amount of L-Trp binding observed with Ca2+ may reflect the different L-Trp binding affinity and kinetics exhibited by the inactive and active receptors. We have modified our text to include these explanations and to indicate that additional studies would be needed to characterize the role of L-amino acid binding to the orthosteric agonist site of CaSR.

Funding

National Institute of General Medical Sciences (R01GM112973)

American Heart Association (15GRNT25420002)

Qing R Fan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank WA Hendrickson and RS Kass for advice and support, Q Liu and O Clarke for advice on diffraction data analysis, C Karan and R Realubit for access to EnVision plate reader, K Rajashankar and Northeastern Collaborative Access Team (NE-CAT) staff for help with data collection. The beamlines at NE-CAT are funded by National Institute of Health (NIH) grants P41 GM103403 and S10 RR029205. This work was supported by American Heart Association grant 15GRNT25420002, and NIH grant R01GM112973 (both to QRF). QRF is an Irma Hirschl Career Scientist, Pew Scholar, McKnight Scholar and Schaefer Scholar.

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